The immunoglobulin molecule consists of one or more sets of four polypeptide chains, two heavy chains having a molecular weight of about 53,000 daltons and two light chains having a molecular weight of about 22,000 daltons, joined by disulfide bonds.
The immunoglobulins are generally subdivided into five classes: G, M, A, D and E. Immunoglobulins of these classes are generally represented by IgG, IgM, IgA, IgD and IgE, respectively.
Five classes of immunoglobulin ar distinguished by the presence of heavy-chain antigenic determinants, which are designated by the lower case Greek character corresponding to the Roman letters applied to the immunoglobulins:
______________________________________ HEAVY-CHAIN ANTIGENIC IMMUNOGLOBULIN DETERMINANT ______________________________________ IgG .gamma. (gamma) IgA .alpha. (alpha) IgM .mu. (mu) IgD .delta. (delta) IgE .epsilon. (epsilon) ______________________________________
There are also subclasses of IgG, IgA, and IgM, based upon other antigenic determinants, which are designated by numerals (e.g., .gamma.1, .alpha.1, .mu.1). Four subclasses for IgG have been recognized, two of IgA, and two of IgM. All subclasses are found in the sera of all normal individuals.
IgG is the most abundant immunoglobulin in the serum of normal humans. It is also found in the tissue fluids, and it can cross the placenta from the material to the fetal circulations. It has antibacterial, antiviral, and antitoxic activities in vivo, and in vitro. It is a late responding antibody.
IgM is characterized by possession of heavy chains with the amino acid sequence that defines the antigenic determinant .mu.. A distinguishing feature of IgM function is its strong cytolytic and complement-fixing activity, which far exceeds that of IgG. IgM is usually the first antibody to appear in animals or humans following immunization. It is then gradually replaced by IgG.
IgA is the second most abundant immunoglobulin in human serum and is the chief secretory immunoglobulin. It is useful in antibacterial and respiratory viral defense.
IgE is the least abundant immunoglobulin. It has skin sensitizing properties and is responsible for a variety of bronchial, gastrointestinal, skin and other allergic reactions.
Very little is known about the structure and biological function of IgD. IgD antibodies have been found in auto-immine diseases and patients sensitive to cow's milk and patients with systemic lupus erythematosus.
Methods for isolating the above immuniglobulin classes are well-known. Methods for raising antibodies to immunoglobulins are also known and methods for binding immunoglobulins to solid supports are known. Methods for isolating antibodies, labeling antibodies with fluorescent molecules, radioactive molecules or enzymes to permit measuring bound antibody are well-known. There are likewise, indirect methods for measuring bound antibody, such as reacting the bound antibody with a labeled (radioactive, fluorescent, enzyme) antibody specific to the antibody.
The determination of antigen specific immunoglobulins of a peculiar class is of particular clinical significance. U.S. Pat. No. 4,020,151 describes an immunoassay for IgG, IgA, and IgM concentrations in the serum which comprises first reacting a solid support with test sample to absorb IgG, IgA, IgM and then reacting a labeled antibody to IgG, IgA and IgM and measuring the bound antibody. Antigen specific immunoglobulins may also be determined utilizing immunoassay techniques, employing immuno components having binding affinity to the antibody to be determined and/or detected. According to such techniques, an immuno component with binding affinity to the antibody to be determined is coupled to a solid support and another specific immuno component is labeled, for example, with a fluorescent, chromophoric, radioactive group or with an enzyme.
However, one disadvantage associated with such techniques is, that if rheumatoid factor, which may be found in serum, is present in the sample false positive results may be obtained. Rheumatoid factor (RF) generally has affinity for antibodies of the IgG class. RF binds via the constant regions of the heavy chains of the IgG molecule. For example, in an assay for a rubella virus class specific immunoglobulin of IgM class, the rheumatoid factor is itself also an immunoglobulin of the IgM class. Because the rheumatoid factor is usually of the IgM class, it will also be bound by anti-IgM immunoglobulins, thereby, producing false positive results. One method for avoiding such rheumatoid factor interference requires separation of the different immunoglobulin class antibodies prior to analysis. However, the methods for separation of immunoglobulins of different classes and especially of antigen specific immunoglobulins of different classes are generally elaborate and time consuming. Such methods include chromatography, electrophoresis, and density gradient configuration.
U.S. Pat. No. 4,273,756 describes an immunoassay for class specific antibodies, IgG, IgM, IgA, IgE and IgD, which comprises first reacting a solid support on which is coated on antibody for a specific immunoglobulin class is contacted with a sample containing a class specific antibody and then reacting the resulting complex with a labeled antigen and measuring the bound labeled antigen.
U.S. Pat. No. 4,292,403 describes an immunoassay for the determination of an antigen specific immunoglobulin of the class IgM, IgA, IgD, and IgE, which comprises contacting the particular antigen specific immunoglobulin with an insolubilized antibody against the antigen specific immunoglobulin or an antigen binding fragment of this anti-immunoglobulin, then treating the mixture with an antigen for which the immunoglobulin has specific affinity and finally treating the mixture with a labeled antigen binding fragment of an antibody against the antigen and then measuring the labeling fragment. This reference suggests that rheumatoid factor interference is removed.
Voller, et al., British Journal of Experimental Pathology, (1975) 56, 338, Gravell, et al., The Journal of Infectious Disease, Vol. 136, Supplement (1977), 5300, and Cleary, et al., Research Communication in Chemical Pathology and Pharmacology, Vol. 19, No. 2, (1978) 281, describe enzyme-linked immunoassays for detecting antibodies to rubella virus.